Project Summary 3D biomimetic tissue engineered scaffold technology is emerging as a promising clinical strategy to repair osteochondral defects within the knee joint. However, regeneration of articular cartilage and subchondral bone with current and promising technology has been limited in scope. Therefore, it would be advantageous to enhance the regeneration using biomaterial, stem cell, and biomolecular technology. The long-range goal of our laboratory is to regenerate complex tissues. One of our immediate objectives is to design biomaterial scaffolds to guide the regeneration of osteochondral tissues. The aim of the proposed study is to evaluate the capability of a porous, sintered PLGA microsphere scaffold delivery system to regenerate subchondral bone. This engineered graft would provide the underlying support necessary to aid in the repair of articular cartilage. In our preliminary work, the small molecule, cAMP, has been demonstrated to enhance the cell adhesion, proliferation, and differentiation of osteoprogenitor MC3T3-E1 cells. However, this small molecule has yet to be evaluated for its capability to induce osteogenic differentiation of MSCs when paired with a biocompatiable, scaffold delivery system. Therefore, we hypothesize that by loading the cAMP small molecule into the porous scaffold, the rate of diffusion will allow for the localized treatment necessary to stimulate bone regeneration. The experimental plan will investigate the cellular response of cAMP/PKA small molecule stimulants on the 3D biomimetic scaffold in three specific aims. The first aim is designed to explore the osteoinductivity of cAMP elevating agents and analogues on rMSCs. Human recombinant BMP-2 will be used as the positive control to evaluate the osteogenic potential. The second aim is designed to develop a 3D cAMP-loaded, porous, PLGA microsphere scaffold and test its bioactivity in vitro. Empty PLGA microsphere scaffolds will serve as the control group. The third aim is to access the biological performance of the engineered scaffold in vivo through a rabbit ulnar critical size defect model. The proposed study will help elucidate the feasibility of developing a cell instructive regenerative biomaterial based on cAMP/PKA stimulated small molecule delivery.